The clinical use of the quinone-containing chemotherapeutic agent adriamycin is restricted by a severe dose-dependent cardiotoxicity. The mechanism underlying cytotoxicity has been linked to an increased generation of superoxide. The NAD(P)H-dependent enzymatic one-electron reduction of adriamycin generates the adriamycin semiquinone radical which undergoes redox-cycling in the presence of oxygen to generate superoxide and adriamycin. Thereby adriamycin provides a kinetic mechanism for one-electron reduction of oxygen by NAD(P)H. Here we show that endothelial nitric oxide synthase (eNOS) stimulates the generation of superoxide from adriamycin. Adriamycin binds to eNOS with a Km of approx. 5.0 micromole as determined from NADPH consumption and ESR spin-trapping of superoxide. Superoxide generation from eNOS/adriamycin is not dependent on the presence of CA(2+)/CaM and is abolished by the flavoprotein inhibitor dephenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. One of the consequences of eNOS-mediated reductive activation of adriamycin is decreased production of nitric oxide and increased superoxide formation. This may lead eNOS to generate peroxynitrite, a potent oxidant implicated in several vascular pathologies.